Recent advances in AAV vectorology have allowed recombinant AAV (rAAV) vectors to be used for human clinical trials. However, the basic biology of AAV vectors is still not well understood. This has been a major limitation for the full exploitation of the usefulness of rAAV vectors. Despite numerous research activities focusing on the tissue tropism and applications for rAAV vectors, the fate of rAAV genomes in vivo and the mechanisms of AAV genome conversion have not been well characterized. Preclinical clinical studies suggest that a disturbing dose of 1x1014 vector genomes may be necessary for a human subject. Our hypothesis is that rAAV genome instability seriously reduces its efficiency. The single stranded (ss) AAV genome can be recognized by host cells as DNA damage signal which leads to a cascade of AAV genome loss/degradation immediately after uncoating. Such mechanism will not allow substantial free ss AAV to exist in the host for an extended period. The limiting step for efficient rAAV transduction is therefore more likely to be intracellular processing of AAV virions and both ss and double stranded AAV genome loss/degradation. In addition, the random integration of AAV genomes is a major concern for AAV vectors. To address this issue, we plan to quantify rAAV integration frequency. Hence, our specific aims are 1). To study the conversion of AAV genome from single stranded DNA to double stranded form. 2). To study the stability of double stranded AAV genomes 3). To quantify the frequency of rAAV integration in vivo. The successful execution of these specific aims will help identify new strategies for improving rAAV transduction efficiency and utilizing rAAV vectors safely. [unreadable] [unreadable] [unreadable]